Shiga toxin (Stx) and ricin, produced by certain E. coli strains and the plant Ricin communicus, respectively, are important causes of human disease and are potential agents of biowarfare. Therapeutic options for persons exposed to these toxins are limited. In particular, antibiotics have no role following ricin exposure and are contraindicated in patients exposed to Stx-producing E. coli. Similarly, there is no specific therapeutics for their inactivation, nor for inhibition of the cell death caused by these toxins. Our laboratory has recently undertaken a screen for small molecules that inhibit intracellular transport of shiga, ricin, and cholera toxins. We have identified several compounds that block transport of these toxins within host cells. These compounds are useful probes of the toxin transport pathway and some may hold potential as therapeutic agents. In tandem with the above screen, which is a cell-based assay and targets toxin transport, we have developed a biochemical assay for toxin enzymatic activity. This assay is highly sensitive and reproducible and has been adapted to high-throughput format. Through the Molecular Libraries Probe Production Centers Network (MLPCN), we propose to screen up to 200,000 compounds for their ability to inhibit Stx and ricin enzymatic activity. Each of these compounds will be verified in a secondary assay then characterized with respect to their potency, specificity, cytotoxicity, reversibility, and effect on related toxins. In future studies, in collaboration with Southern Research Institutes, compounds with greatest therapeutic potential will be subjected to structure-activity relationship (SAR)-guided optimization of potency, specificity, lack of toxicity, and drug-like properties. These will be tested in an animal model of toxin-mediated disease. PUBLIC HEALTH RELEVANCE: This work has major implications for public health. Stx causes several hundred thousand cases of colitis in the US annually and accounts for thousands of deaths. In the event of their intentional release as bioweapons, these toxins could significance morbidity and mortality. We propose to identify and optimize as these compounds as the first drugs that could be used to prevent or treat individuals exposed to these toxins.